Distribution and risk factors associated with intestinal parasite infections among children with gastrointestinal disorders.

AIM
Prevalence and risk factors associated with intestinal parasites among children ≤ 12 years old in Nahavand county western Iran, was the objective of this search.


BACKGROUND
Intestinal parasites (IPs) are important health problems among most societies.


METHODS
This cross sectional study was carried out during 6 months from April to September 2014 in Nahavand County western Iran. Fecal samples were collected from 500 children suffering from gastrointestinal disorders (GIDs) and examined by macroscopy and microscopic (using saline and iodine wet mount, formalin-ether sedimentation, Trichrome and modified Ziehl Neelsen staining) methods. Finally, data was analyzed using Chi-square (Chi2) test and Fisher's exact test as well as logistic regression.


RESULTS
21.8% (109/500) of the samples were infected by one or more IPs. The most common parasites were Blastocystis sp. (16.2%), followed by Cryptosporidium spp. (2.6%), Giardia lamblia (1.6%), and Entamoeba coli (1.6%). Prevalence of intestinal parasite infections were significantly associated with age (OR= 2.280; CI 95% = 1.375-3.830; P<0.002), gender (OR= 0551; CI 95% = 0.348-0.875; P<0.011), contact with domestic animal or soil (OR= 0.492; CI 95% = 0.282-0.860; P<0.013) and seasons (OR= 2.012; CI 95% = 1.254-3.227; P<0.004). There was a significant correlation between IPs with diarrhea (OR= 3.027; CI 95% = 1.712-5.345; P<0.001) and nausea or vomiting (OR= 3.261; CI 95% = 1.281-8.175; P<0.013).


CONCLUSION
Blastocystis sp. was the most prevalent parasites among children in Nahavand County and Helminthes infection have been dramatically decreased. Our finding shown that gender, age, season and contact with domestic animals or soil polluted are main predictive factors for intestinal parasite infections among children in this region. Moreover, IPs infection among children with gastrointestinal disorders were significantly associated with diarrhea and vomiting or nausea signs.


Introduction
threatened/or unthreatened water, hand washing practices before meal and after the toilet and etc (16)(17)(18)(19). The epidemiological data based on the frequency of IPIs is one of the basics for developing appropriate control measures. Previous epidemiological studies in Iran have demonstrated high prevalence of IPIs among different population and have shown that Blastocystis sp., Entamoeba histolitica/E. dispar, Giardia lamblia, Cryptosporidium spp., Enterobius vermicularis and Ascaris lumbericoides are the predominant IPIs (1,2,15,(20)(21)(22)(23)(24)(25). Surprisingly, in spite of the public health importance of these infections and the potential consequences, there was no enough data about their epidemiology of IPIs among children in western Iran. The aims of this study were to identify the epidemiology and associated risk factors of IPIs in children with gastrointestinal disorders in Nahavand County, western Iran.

Study areas & population:
This cross-sectional survey was carried out from April to September 2014 in Nahavand County, Hamadan province, western Iran. Stool samples from 500 children under 12 years old suffered from GIDs who referred to medical centers laboratories were randomly collected. The sample size was calculated using formula: n = z 2 *p(1-p)/d 2 under assumptions as follow: the reference prevalence was considered as 35 % (20), a 95% level of confidence and 5% marginal error. The sample size required was 496, but for more accuracy 500 samples were collected.

Questionnaire
The questionnaire was prepared to elicited information on the demographic data (age, gender and location (urban & rural)), acute clinical symptoms (diarrhea and dysentery, abdominal pain, stomach pain, bloating, vomiting & nausea), environmental sanitation and living condition characteristics (season, contact with domestic animals and soil) which will be used to assess the potential risk factors for IPIs. Informed consent was obtained from each attendee prior to the sampling. Certain criteria were applied to enroll children in the study. Inclusion criteria included age ≤12 years old with no history of taken anti parasites drugs in two weeks before the test. Exclusion criteria included children suffering from GIDs (diarrhea, dysentery diarrhea, stomach pain, bloating and Nausea or vomiting) disease.

Stool Collection and Processing
From all of the participants a single fresh stool sample were collected from 12 clinical laboratories in Nahavand County. The collected specimens were transported to the parasitological laboratory of Ayatollah Alimoradian hospital, in Nahavand city for the stool analysis process. At first, Information related to patients was recorded on a daily basis in the registered office information and stool samples were observed macroscopically to consistency and the presence of worm, larva, segments, blood, and mucus were recorded.
After macroscopic analysis, direct wet mount with normal saline (0.85% NaCl solution) in all of the stool specimens in one side of the slides (for the presence of motile intestinal parasites and trophozoite) was done and lugol's iodine staining was performed to distinguish cysts of intestinal parasites in other side of the slide. In the next step formalinether concentration was prepared and the sediments were stained with iodine, put on a slide and covered with a cover slip to accurate detection of cyst or eggs of intestinal parasites. Moreover, slide smear was prepared in fresh stool and immediately stained with trichrome's staining to accurate differentiation of intestinal protozoa (Entamoeba, Giardia, Blastocystis sp. and etc). For detection of coccidian parasites (Cryptosporidium spp.), Modified Zeihl-Neelsen staining after concentration technique was done. All of the smears prepared were microscopically observed using 100x, 400x, and1000 x magnification (26).

Statistical analysis
For statistical analysis the data was exported to Statistical Package for the Social Sciences software version16 (SPSS, Chicago, IL, USA). The proportion percentage was used to describe the characteristics of the participants, including the frequency of IPIs according to age, sex and etc. A Pearson's Chi-square (Chi2) test and Fisher's exact test were used for differences in the proportions of IPIs between different variables and odds ratios (OR) and 95% confidence intervals (CI) were used for associations of variables. The t-test wasused to compare the mean age. Logistic regression was used to detect risk factors of parasite infection. The P value <0.05 was considered statistically significant.

Ethical clearance
The all the procedures of this study were approved by the Ethics Committee of the Shahid Beheshti University of Medical Science (SBMU). Iran, before the beginning of the study. All study participants were informed about the study procedures and written informed consents were obtained from all of them prior to sample collection.

Sociodemographic characteristic
Out of 500 children with GIDs, 278 boys and 222 girls, aged 22 days-12 years were enrolled in this study. 246 children lived in rural areas and 254 subjects lived in urban area.149 of 500 participants had frequently contact with domestic animal or soil and 351 child no contact. The proportion of children according to age groups were ≤ 3years (276 patients), 4 -6 years (135 children) and 7 -12 years (89 isolates). These specimens were taken during spring (224 n: number; * The frequency of intestinal protozoa was significantly higher than intestinal helminthes.

Risk factors and Clinical features associated with (IPIs)
Logistic regression by forward method detected the risk factors associated with IPIs among children with GIDs and socio-demographic, environmental and personal hygiene factors evaluated in this study(

Discussion
Epidemiological studies based on frequency of IPs infection in different society, have primary objective to identify high-OR: odds ratio; n: Number; Reference: The top group is considered as baseline; *Chi 2 as well as Fisher exact test are used. Also, univariate Logistic regression was used for age group analysis *p< 0.05  (21), and is very higher than checkup individuals under 15 years old (9.3%) in Nahavand laboratories (21). 21.6% of children infected by one or more protozoan parasites and only one children infected by helminthes (Enterobius vermicularis) infection. However prevalence of helminthes infection may be increased if cellophane tape, The Baerman techniques and agar plate culture were used (27). However, our study and recently reported studies in different part of Iran (28), Isfahan (29), Hamadan (30), Nahavand (21) represent a dramatic decrease of helminthic infections (Ascariasis, hook worms, Entrobiosis and etc.) and protozoan infections (Ameobiasis, Giardiasis, Cryptosporidiosis and etc.). It may be results from increasing public and individual hygienic, increase awareness of the prevention and control of parasitic infections and access to the safe food and water sources. Also, variations in prevalence of IPs may be due to differences in climatic conditions, environmental hygiene, economic and educational status and study subjects, and previous control efforts.
The most common IPs in our study was Blastocystis sp. with 81 cases (16.2%). It is similar to previous studied in Nahavand County. This study and other study in Tehran and Isfahan show that prevalence of Blastocystis sp. recently increased in Iran (21,29,31). But the pathogenicity of Blastocystis sp. is still controversial, some studies suggest that it is a pathogen (32,33), whereas other studies believe it is a non-pathogen or commensal (33,34). Recently, discussion on pathogenesis and accurate diagnosis from Blastocystis sp. can be cause of increase reported of this parasite. In our study Blastocystis sp. are related with diarrhea and season. Also previous clinical studies by Tan et al. and Moosavi et al. presented that abdominal pain and diarrhea are two the major signs among Blastocystis-positive patients (31,32). Regardless of Blastocystis sp., Cryptosporidium spp. and Giardia lamblia with the frequency of 13 (2.6%) and 8 (1.6%) respectivelly was the most common intestinal pathogenic protozoan. Because, staining method for Cryptosporidium spp. and concenteration methods are not usulally use in laboratories , Modified Zeihl-Neelsen staining after concentration technique specifically in children with GIDs is required in this region and other same regions.
In this study, Cryptosporidium spp., Giardia lamblia and Blastocystis sp. have the most prevalent IPIs in children with GIDs. Frequency of Cryptosporidium spp., Giardia lamblia and Blastocystis sp. with Socio-demographic and clinical features in children with GIDs are shown in Table 4. There was significant relationship between cryptosporidiosis by age (p = 0.023), location (p= 0.021), contact with domestic animal and soil polluted by animal feces (p= 0.001), season (p= 0.045), vomiting/ nausea (p= 0.001) and diarrhea (p= 0.031). Significantly different observed among Giardiasis by age (p= 0.001) and contact with domestic animal or soil (p= 0.010).There was significant correlation among Blastocystis sp. by age (p= 0.009), season (p= 0.017) and diarrhea sign (p= 0.007).
Blastocystis sp.,Cryptosporidium spp. and Giardia lamblia are zoonotic protozoa that are a great threat to public health and can be transmitted through contaminated water and food. Water and food contaminated with animal wastes and farming practice are important source of transmission. Intestinal parasitic infections, especially Cryptosporidium spp. and Giardia lamblia were significantly associated with contact with domestic animals and soil. Thus it is necessary that animals be kept away from water and food source that are used by human. Animal husbandry and agriculture are common in Nahavand County, in the warm season livestock going to pastures and contamination of water sources and vegetables by livestock fecal can increase pollution. Also frequency of IPs (especially Cryptosporidium spp. and Blastocystis sp.) in warm season (summer) is higher than cold season (spring).
According to age groups significant different was observed among children 6-12 years old by Cryptosporidiosis and giardiasis. It may be due to contact children with polluted source such as vegetable in farms or soil polluted by animal feces during play in soil land and farms especially in rural region or direct contact (person to person) during meal food or etc. together.
In relation to poly parasitism, our results are similar to previous study in Isfahan, Hamadan and previous study in Nahavand County that poly parasitism detected. The observed co-infection could be clarified by the facts that many species of protozoa (Cryptosporidium spp., E. histolytica/E. dispar, Blastosystis sp., Entamoeba coli, Giardia lamblia and etc.) have the same mode of transmission and that hygiene is poor in these areas (21,29,30).
Infection happen by IPs may be consequences such as gastrointestinal disorders (diarrhea or dysentery and etc., anemia, malabsorption, delay growth in children and physical complications, therefore, it was seen as the main health problem (31,35,36). According to clinical signs in our study there was significant correlation among IPs and cryptosporidiosis in GIDs children with diarrhea, and vomiting/ or nausea. Similar to our results various studies in other countries and in severe acute malnutrition related diarrhoea have shown intestinal parasites as risk factors of diarrhoea (37,38 (2, 31 ,39). Also in turkey the most common complaint related with IPIs was intestinal dysmotility, nausea/ vomiting and abdominal distention (8).
There were some limitations in our study. 1. Stool samples were collected only once, It is likely that the analysis of three consecutive stool samples could have increased the diagnostic sensitivity.
2. Sample collected was done during two warm season and there is no data about autumn and winter.
3. Due to the lack of facilities and financial funding we could not use all the laboratory available techniques, such as the Baerman technique to find larvae of helminth including Strongyloides stercoralis or cellophane tape to detect Enterobius vermicularies and agar plate culture to find accurate detection of helminthes larva. 4. Also, it is likely that the prevalence of some protozoa such as Entamoeba and Blastocystis sp. could be even higher if culture techniques had been used.
Based on the present study Blastocystis sp. is the prevalent parasites in Nahavand County. Especially pathogenic parasites such as Cryptosporidium spp. and Giardia lamblia among children with GIDs, still are prevalent in western Iran, and helminthes infection have been dramatically decreased during the past decades. Thus, Modified Zeihl-Neelsen staining after concentration technique is recommended for Cryptosporidium spp. identification in this area. Also, our finding suggests the risk factors such as gender, season and contact with domestic animals and soil polluted are increase susceptibility of children with IPs infection. IPs infection among children with GIDs are associated with diarrhea and vomiting/or nausea in this area and the role of parasites in children's should be considered. Thus, health interventions to children and prevent children from contact with contaminated resources such as land and livestock waste in this area is necessary.